Biochemical Profiles of the Hemolymph of the Horseshoe Crab (Limulus polyphemus)
IAAAM Archive
Stephen A. Smith1; Daniel Boon1; Tanya L. Tag1; James M. Berkson2
1Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA; 2Department of Fisheries and Wildlife, Virginia Polytechnic Institute and State University, Blacksburg, VA


The horseshoe crab (Limulus polyphemus) is one of four extant species remaining worldwide, and can be found along the western Atlantic coast from Maine to the Gulf of Mexico. Demand for this species of horseshoe crab continues to grow. Horseshoe crab eggs are an important protein source to the successful spring migration of at least eleven species of migratory shorebirds, a component of the horseshoe crab's hemolymph (Limulus Amoebocyte Lysate or LAL) is used worldwide to detect the presence of endotoxins in injectable drugs and implantable medical devices4,5 and horseshoe crabs are also used as bait for a growing eel and conch fisheries along the east coast of the United States. In addition, horseshoe crabs can be found as inhabitants in most "touch tanks" at public aquaria. Thus, horseshoe crabs are an essential component of a healthy coastal ecosystem, an essential contributor to human health, and an integral part of the coastal economies of the Eastern United States. Unfortunately, the status of wild horseshoe crab populations may be in jeopardy due to over harvesting, environmental degradation and habitat destruction.1,2,3

While the horseshoe crab has existed in its present form for over 350 million years virtually unchanged, very little is known about the basic biology of this organism. Most studies have focused on the horseshoe crab's life history, natural history, and unique vision and neuroanatomy.6 While the unusual characteristics of the crab's "blue blood" and the composition of the LAL have been studied in detail, basic information on the horseshoe drab's physiological parameter are conspicuously lacking. Thus, this project attempted to fill part of this void by examining the biochemical parameters of the hemolymph of adult horseshoe crabs.

Fifty adult (29 male and 21 female) horseshoe crabs were collected during the month of September off the coast of Ocean City, MD. Approximately 10-12 ml of "blood" was collected from each crab with a sterile 14-gauge trocar needle, and transported on ice to the laboratory where the cells were removed by centrifugation at 150.0 x g for ten minutes. The resulting hemolymph was then removed and stored at-20°C until analysis.

Hemolymph samples were analyzed using an automated dry chemistry system (Kodak Ektachem 700, Eastman Kodak Co., Rochester, NY, USA) for total protein, glucose, creatinine, cholesterol, sodium, potassium, chloride, Calcium, magnesium, and phosphorus concentrations, triglycerides, amylase, lipase, and alkaline phosphatase (ALP) and aspartate aminotransferase (AST) and gamma glutamyl transferase (GGT) activities. Initially albumin, globulin, direct and indirect bilirubin, urea nitrogen, and creatine kinase (CK) were also evaluated, but were eventually deemed inappropriate due to extremely low or non-existent levels. Copper levels in the hemolymph were determined by atomic absorption using a spectrophotometer (Beckman Du 680 B, Fullerton, CA, USA) set at 580A, while hemolymph osmolality was determined using a freezing point depression osmometer (Advanced Micro-osmometer, Model 3 MoPlus, Advanced Instruments, Inc, Norwood, MA, USA).

Results of the biochemistry parameters (mean values) for the hemolymph of the horseshoe crab were: total protein (8.15 g/dl), glucose (58.5 mg/dl), creatinine (0.7 mg/dl), cholesterol (0.8 mg/dl), sodium (389.5 mEq/1), potassium (12.5 mEq/1), chloride (445.1 mEq/1), calcium (39.0 mg/dl), magnesium (96.1 mg/dl), phosphorus (3.4 mg/dl), triglycerides (5.3 mg/dl), amylase (9.3 U/l)), lipase (32.7 U/l), alkaline phosphatase (12.1 U/l), aspartate aminotransferase (5.4 U/l) and gamma glutamyl transferase (0.92 U/l).


1.  Berkson, J. M., and C. N. Shuster, Jr. 1999. The horseshoe crab: The battle over a true multiple-use resource. Fisheries 24(11).

2.  HCTC. 1998. Status of the Horseshoe crab (Limulus polyphemus) population of the Atlantic coast. Horseshoe Crab Technical Committee. Atlantic States Marine Fisheries Commission, Washington, D.C.

3.  Manion, M. M., R. A. West, and R. E. Unsworth. 2000. Economic assessment of the Atlantic coast horseshoe crab fishery. Prepared for the Division of Economics, U.S. Fish and Wildlife Service, Arlington, VA. by Industrial Economics, Inc., Cambridge, MA.

4.  Mikkelsen, T. 1988. The secret in the blue blood. Science Press. Beijing, China. 125 pgs.

5.  Novitsky, T. J. 1984. Discovery to commercialization: the blood of the horseshoe crab. Oceanus 27(1 ): 13-18.

6.  Shuster, C. N., Jr. 1982. A pictorial review of the natural history and ecology of the horseshoe crab, Limulus polyphemus, with reference to other limulidae. Pages 1-52 in J. Bonaventura, C. Bonaventura, and S. Tesh, eds. Physiology and biology of horseshoe crabs: studies on normal and environmentally stressed animals. Alan R. Liss, Inc., New York.

Speaker Information
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Stephen A. Smith, DVM, PhD
Department of Biomedical Sciences and Pathobiology
Virginia-Maryland Regional College of Veterinary Medicine
Virginia Polytechnic Institute and State University Phase II
Blacksburg, VA, USA

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